egfp-ip 3 r1 construct  (Absolute Biotech Inc)

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: Endogenous IP 3 R1s form puncta. a In-gel fluorescence of lysates from EGFP-IP 3 R1 HeLa cells (GR) and control (WT) cells demonstrates that the only fluorescence is associated with EGFP-IP 3 R1 (green arrow). Results typical of four gels. Positions of selected M r markers (kDa) are shown ( a , c , d ). b TIRFM images of EGFP-IP 3 R1 HeLa cells showing a marker for the ER lumen (mCherry-ER). The merged image and an enlargement of the boxed area show co-localization of EGFP-IP 3 R1 with mCherry-ER (Pearson’s coefficient with Costes’ automatic threshold = 0.93 ± 0.02; Costes P value = 1.00, n = 4 cells). Scale bar = 5 µm (2 µm for enlargement). c Western blots (WBs) for IP 3 R1-3 show expression of tagged (green arrow, ~290 kDa) and untagged (black arrow, ~260 kDa) IP 3 R1 in GR and WT cells, respectively. Expression of IP 3 R subtypes in GR cells is shown relative to control (WT) cells (%, mean ± SD, n = 3 for IP 3 R2 and IP 3 R3, n = 4 for IP 3 R1). Comparisons of band intensities using paired Student’s t -tests indicated no significant differences between WT and EGFP-IP 3 R1 cells. d WB (IP 3 R1-3 antibodies) from lysates of EGFP-IP 3 R1 HeLa cells after immunoprecipitation with GFP-Trap. Eluate lanes were loaded with sample equivalent to 1.5 times the amounts loaded in the lysate lanes. Numbers show % of each subtype detected in the pull-down ( n = 2). e Photobleaching of a punctum showing the final bleaching step (bracket) and the initial fluorescence (dashed line) used to calculate the total number of fluorophores ( n ). FU, fluorescence units. f Single-step photobleaching results (284 puncta from five cells, Supplementary Fig. ) were used to calculate the number of tetrameric IP 3 Rs per punctum (8.4 ± 7)

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Fluorescence, Marker, Western Blot, Expressing, Immunoprecipitation

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: IP 3 Rs form mobile and immobile puncta. a Time-lapse TIRFM images (0.6-s intervals) of EGFP-IP 3 R1 in cells expressing mCherry-ER. Track of a single particle, with the first and last positions shown by white and yellow arrows, respectively. Scale bar = 5 µm. b Representative epifluorescence image of an EGFP-IP 3 R1 HeLa cell with perinuclear (blue) and peripheral (magenta) regions highlighted for FRAP analysis (circular bleached area, radius = 1.84 μm). The boxed area is enlarged to show pre- and post-bleach (after 120 s) images of the peripheral region. Scale bars = 5 µm. c Normalized fluorescence intensities recorded from peripheral or perinuclear regions in a typical FRAP experiment with live and fixed EGFP-IP 3 R1 HeLa cells. d , e Summary results show mobile fractions ( M f , mean ± SEM) ( d ) and diffusion coefficients ( D , mean and all values) ( e ) for perinuclear (25 cells) and peripheral regions (26 cells). **** P < 0.0001, * P < 0.05, two-tailed Student’s t -test. f Distribution of fluorescence intensities for individual mobile and immobile puncta. Inset shows distribution for the brightest immobile puncta. g Relative numbers of mobile and immobile puncta, and distribution of fluorescence between them (%). Results ( f , g ) are from time-lapse TIRFM images of 10 cells (mean ± SD). h , i Analysis of bleaching steps of brightest and dimmest puncta in fixed cells was used to determine the step amplitude ( h ) and number of steps ( i ) for each punctum (Supplementary Fig. ). Mean ± SD, with 23–25 puncta analysed in each of two cells. *** P < 0.001, **** P < 0.0001, Student’s t -test

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Expressing, Single Particle, Fluorescence, Diffusion-based Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: IP 3 Rs within mobile puncta do not exchange with immobile puncta. a TIRFM images of FRAP experiment show images before, immediately after and 60 min after bleaching. Bleached area shown by white rectangle. Times shown as h:min:s. b , c Enlarged images of yellow boxed area in a . Immobile puncta were identified by overlaying two frames (30 s apart) using pseudocolours for each (green and magenta), such that immobile puncta appear white in the overlay (iii, iv) (Supplementary Fig. ). Images were captured before photobleaching ( b ) and after recovery for 15 min ( c ). Enlarged areas (red boxes in iii) are shown in (iv). Scale bars ( a – c ) = 10 µm. d Enlargements (blue boxes in b iv and c iv) show images before and 15 min after bleaching. Scale bar = 2 µm. Abundant green and magenta puncta in the post-bleach images ( c , and lower in d ) indicate rapid exchange of mobile EGFP-IP 3 R, while the scarcity of white puncta suggests very slow exchange of immobile EGFP-IP 3 R1. e Summary results show fluorescence recovery ( F / F 0 , %, mean ± SEM) from three cells (26 ± 15 immobile puncta per cell were identified in the initial images). f Example trace from a region centred on an immobile punctum (circled in first image) shows stepwise increase and decrease in fluorescence intensity as a mobile punctum (arrows) moves through the immobile punctum (Supplementary Movie ). Scale bar = 1 μm

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Fluorescence

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: EGFP-IP 3 Rs are diffusely distributed within puncta. a Examples of TIRFM and subsequent STORM images of EGFP-IP 3 R1 puncta. Scale bar = 0.5 µm. b , c Before STORM, mobile and immobile puncta were identified using TIRFM and by overlaying pseudocoloured images collected at 10-s intervals (Supplementary Fig. ). Examples of TIRFM and STORM images show immobile ( b ) and mobile puncta ( c ). Overlays show that EGFP-IP 3 R1s are diffusively distributed within mobile and immobile puncta. Scale bars = 100 nm. The yellow squares show approximate dimensions of an IP 3 R (20 nm × 20 nm). d Analysis of STORM images shows the area enclosing all EGFP-IP 3 R1s within each punctum and the density of localization events within the punctum for mobile and immobile puncta (mean ± SD for 4–6 puncta). * P < 0.05, Student’s t -test, relative to immobile puncta. Results show that mobile puncta are smaller and contain fewer IP 3 Rs than immobile puncta. e Many IP 3 Rs within puncta are too diffusively distributed to allow direct interactions between them. Although we used a non-oligomerizing EGFP to tag IP 3 R1 and minimize potential artefacts, the diffuse spacing of some EGFP-IP 3 R1s confirms that puncta are not formed by interactions between EGFP

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques:

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: IP 3 Rs move by diffusion and by directed motion along microtubules. a TIRFM image used for single-particle tracking shows characteristic reticular ER. Scale bar = 5 µm. b Trajectories of all puncta from the region defined by white lines in a . Colours indicate different trajectories. c Enlargement showing trajectories within the boxed area in a . d Average mean squared displacement (MSD) for all puncta within each cell plotted against time. Results are from five cells, with 225–926 puncta analysed in each. e Pooled results from five cells (2,698 tracks). f Examples of single-particle trajectories (collected at 100-ms intervals) for an immobile punctum (i) or mobile puncta moving by diffusion (ii) or directionally (iii). g Distribution of EGFP-IP 3 R1 puncta between mobility states. Trajectories of 1,509 puncta from five cells were analyzed by TraJClassifier (Supplementary Fig. ). h Wide-field images, typical of nine cells, show EGFP-IP 3 R1 puncta, microtubules (mCherry-tubulin) and their co-localization. Final panel shows an enlargement of the boxed area in the overlay. Scale bar = 10 µm (2 µm in enlargement)

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Diffusion-based Assay, Single-particle Tracking, Single Particle

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: Ca 2+ puffs evoked by photolysis of caged-IP 3 occur at immobile IP 3 Rs. a TIRFM image of a single cell loaded with EGTA and Cal-590 shows immobile IP 3 Rs (white, see Supplementary Fig. ) and Ca 2+ puffs (red) evoked by photolysis of caged-IP 3 . Boxed area of the overlay (iii) is shown enlarged in (iv). Scale bars = 10 µm. b Co-localization of Ca 2+ release and immobile IP 3 R puncta shown by their fluorescence intensity profiles along the dashed line in a iv. EGFP profiles were captured immediately before and ~30 s after recording Cal-590 fluorescence. c Enlargements of four puff sites highlighted in a show co-localization of each Ca 2+ release event with an immobile IP 3 R punctum. d Enlargements of site 2 (from a ) show four successive puffs generated at the same immobile punctum. Scale bars = 2 μm ( c , d ). e Temporal profiles of Cal-590 fluorescence changes (Δ F / F 0 ) show Ca 2+ puffs last ~200 ms. f Summary shows the fraction of Ca 2+ puffs evoked by histamine (432 puffs in two cells, mean ± range) or photorelease of IP 3 (871 puffs in three cells, mean ± SD) occurring at immobile IP 3 R puncta. Events and puncta were considered to occur at the same site if the centre of mass of the peak change in Cal-590 fluorescence was within 6 pixels (0.96 µm) of the punctum. g Comparison of fluorescence intensity distributions of all detected EGFP-IP 3 R1 puncta, and puncta associated with Ca 2+ puffs evoked by photolysis of caged-IP 3 . Results are from three cells. Dashed lines show 50th and 95th percentiles for the intensities of all puncta

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Fluorescence, Generated

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: Depletion of ER Ca 2+ stores causes native STIM1 to accumulate at functional ER-PM junctions adjacent to immobile IP 3 R puncta. a , b Representative TIRFM images of EGFP-IP 3 R1 HeLa cells fixed and immunostained for STIM1 before ( a ) or after treatment with thapsigargin (Tg, 1 µM, 15 min) to deplete the ER of Ca 2+ ( b ). Overlaid images of Tg-treated cells show no significant co-localization of STIM1 and IP 3 R puncta (Pearson’s coefficient with Costes’ automatic threshold: 0.331 ± 0.026, n = 7 cells). c Distribution of mobile (green and magenta) and immobile (white) IP 3 R puncta in Tg-treated cell. Scale bars ( a – c ) = 10 µm. d Enlargements of the boxed regions in b show that immobile IP 3 R puncta (identified before fixation ( c ), with all shown by arrowheads) abut STIM1 puncta without coinciding with them. Scale bars = 2 µm. e Fluorescence intensity profiles for EGFP-IP 3 R1 and STIM1 across the lines shown in d . Distances (μm) between the peaks of the fluorescence intensity for STIM1 and immobile IP 3 R are shown. f Co-localization of CFP-STIM1 (pseudocoloured green) and mCherry-Orai1 (red) puncta in a Tg-treated HeLa cell. We used tagged proteins because available antibodies do not reliably detect endogenous Orai1. Scale bar = 10 µm (2 μm in enlargement)

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Functional Assay, Fluorescence

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: STIM1 translocates to ER-PM junctions adjacent to immobile IP 3 R puncta. a , b TIRFM images show that thapsigargin (Tg, 1 µM, 5 min) causes formation of STIM1-mCherry puncta at ER-PM junctions (i). Immobile IP 3 Rs (ii, white, identified from overlays of pseudocoloured EGFP-IP 3 R distribution at 30-s intervals) abut STIM1 puncta (iii). Enlarged image of the boxed area shows all immobile EGFP-IP 3 R1 (arrows) and STIM1-mCherry without pseudocolours for clarity (iv). Scale bars = 10 µm (i–iii), 2 µm (iv). c Similar overlay images from three additional Tg-treated cells, with all immobile IP 3 R punta identified with arrows. Scale bars = 2 µm. d Fluorescence intensity profiles for EGFP-IP 3 R1 measured at 30-s intervals (green and magenta) and STIM1-mCherry for transects drawn across Tg-treated cells. Results show that immobile IP 3 Rs (white, where green and magenta coincide) and STIM1 after store depletion are juxtaposed, but not perfectly aligned. Distances (μm) between the peaks of the fluorescence intensity for STIM1 and immobile IP 3 R are shown; 84 ± 9% (mean ± SD, n = 3 cells) of the centroids of immobile EGFP-IP 3 R1 puncta were within twice their radius (2 r = 0.64–0.96 μm) of a STIM1 punctum. e STIM1-mCherry fluorescence was measured before ( F 0 ) and after Tg treatment ( F Tg ) at ROI with twice the radius of underlying mobile or immobile EGFP-IP 3 R1 puncta. Summary results show the change in mCherry fluorescence (Δ F = ( F Tg − F 0 )/ F 0 , mean ± SEM, n = 3 cells, 29–33 puncta). * P < 0.05, Student’s t -test. f , g Density of STIM1-mCherry and EGFP-IP 3 R1 puncta from randomly selected peripheral regions of cells (where puncta are most clearly separated) ( f ) and the separation between any STIM1 puncta identified within the randomly selected regions and the nearest STIM1 or immobile EGFP-IP 3 R1 punctum ( g ). h Frequency distributions for the separations of STIM1 puncta from mobile and immobile EGFP-IP 3 R1 puncta. Whereas 28% of mobile IP 3 R puncta were within 300 nm of a STIM1 punctum, only 10% of immobile IP 3 R puncta fell within this distance. Results ( f – h ) are from four cells. ** P < 0.01, Student’s t -test

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Fluorescence

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: Ca 2+ signals evoked by SOCE occur alongside immobile IP 3 Rs. Clustered IP 3 Rs evoke Ca 2+ puffs when they bind IP 3 and then respond to Ca 2+ released by a neighbouring IP 3 R. Only a small fraction of all IP 3 Rs, namely immobile clusters close to the PM, are licensed to respond (green). The licensed IP 3 Rs are adjacent to the junctions where STIM1 and Orai interact to mediate SOCE, which is itself locally regulated by Ca 2+ passing through Orai (red lines). We suggest that the juxtaposition of responsive IP 3 Rs and the SOCE machinery allows local depletion of ER Ca 2+ stores to activate SOCE, while sparing it from inhibition by the large Ca 2+ fluxes through IP 3 Rs

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Inhibition